UCn2, a 38 amino acid peptide, was discovered in 2001 (1). Studies in animals and human subjects with heart failure (HF) have shown favorable effects of UCn2 peptide infusion, including increased cardiac output (4). However, because of a short half-life (minutes) the peptide’s beneficial effects require continuous i.v. infusion, rendering such therapy impractical except for brief use in hospitalized subjects. We therefore investigated the usefulness of UCn2 therapy via gene transfer, which would circumvent the need for continuous i.v. infusion and enable long-term outpatient therapy.

We recently demonstrated in mice that i.v. delivery of an adeno-associated virus type 8 vector encoding murine UCn2 (AAV8.UCn2) results in increased plasma UCn2 levels within 6 weeks, an effect that persists for at least 7 months without inflammation or unfavorable hemodynamic effects (5). In the course of these experiments, it was noted that fasting blood glucose levels were significantly reduced in normal mice that had received UCn2 gene transfer. We therefore conducted studies in 2 murine models of insulin resistance analogous to clinical type 2 diabetes mellitus (T2DM) testing the hypothesis that a single i.v. injection of AAV8.UCn2 would increase glucose disposal by promoting insulin sensitivity.

UCn2 gene transfer and glucose disposal in HFD model of insulin resistance. To determine whether UCn2 gene transfer increased glucose disposal in the setting of insulin resistance, we used high fat diets (HFDs) to induce insulin resistance in mice. HFD-fed mice that received UCn2 gene transfer showed consistently lower glucose levels. The AUC in glucose-tolerance tests was reduced by 40%–42% (Figure 2A; P = 0.003). Similar responses on glucose disposal were seen whether UCn2 was delivered before or after the onset of hyperglycemia (Figure 2A), confirming a treatment effect on preexisting hyperglycemia.

Metabolic assessment. The Comprehensive Laboratory Animal Monitoring System (CLAMS) was used to assess the effects of UCn2 on metabolic features of HFD-fed mice. No group differences were seen in mean rates of oxygen consumption, carbon dioxide production, activity, or heat generation. HFD-fed mice that received UCn2 gene transfer showed small (2.4%) but statistically significant increases in respiratory exchange ratio (Table 3).

The most important finding of the current study is that UCn2 gene transfer has a profound and enduring effect on insulin sensitivity in HFD-fed mice (Figure 3). A single i.v. injection of AAV8.UCn2: (a) prevents hyperglycemia from developing in mice fed a HFD and increases glucose disposal if delivered after hyperglycemia is present (Figure 2A); (b) increases insulin sensitivity, glucose disposal rate, and glucose uptake in skeletal muscle (Figure 3, B and C, and Figure 6C); (c) reduces fatty infiltration of the liver (Figure 7, B–D); (d) reduces weight gain on prolonged HFD (Figure 7A); and (e) decreases fasting hyperglycemia and increases glucose disposal in a second model of disordered glucose homeostasis (db/db mice) (Figure 5, B and C). These data confirm the efficacy of UCn2 gene transfer in reducing insulin resistance. These results have potential clinical application.

Clinical trials of i.v. infusions of UCn2 peptide (4) or a related peptide, stresscopin (7), in patients with HF have not reported reduced blood glucose levels. Perhaps the effects on glucose metabolism conferred by UCn2 gene transfer are a consequence of longer duration and higher plasma UCn2 concentrations obtained with gene transfer than with UCn2 peptide infusion. Indeed, 6 weeks after UCn2 gene transfer (5 × 1011 gc, i.v.) in mice, mean plasma levels were 15-fold control (5), while peak UCn2 levels in human subjects during 1- to 4-hour peptide infusions (5–30 ng/kg/min) were 3-fold control and short-lived, rapidly declining after termination of the infusion (4). However, it is unlikely that the high plasma UCn2 levels attained in the present study are required for an effect on glucose metabolism. Our dose-response data indicate that AAV8.UCn2 amounts 30-fold lower provide a glucose-lowering effect (Figures 8, A–D), which corresponds to a plasma UCn2 level of 4.0 ± 0.5 ng/ml, 4-fold above normal (Figure 8A).

The present study documents the effects of UCn2 gene transfer on glucose disposal in 2 murine models of insulin resistance. Although the study was primarily conducted in male mice, female db/db mice also showed normalization of fasting glucose after UCn2 gene transfer, suggesting that the effect occurs in both sexes.

Mechanism. Our data show beneficial effects of UCn2 gene transfer on glucose uptake in skeletal muscle in the basal state (Figure 6C) and augmentation of glucose disposal upon insulin stimulation (Figure 3C). Therefore, skeletal muscle clearly contributes to the increased insulin sensitivity observed. UCn2 gene transfer also was associated with a 20% reduction in HGP (Figure 3D), which may have contributed to improved glucose homeostasis.

Insulin acts by increasing Akt phosphorylation of Ser-473, which promotes Glut4 translocation to the plasma membrane and increased glucose disposal. UCn2 peptide increases Glut4 translocation similarly to insulin (Figure 6B), but, in contrast to insulin, phosphorylation of Ser-473-Akt is decreased (Figure 6B), indicating a pathway for UCn2-related Glut4 translocation and increased glucose disposal. Glut4 translocation is associated with AMPK phosphorylation (Figure 6B) (8) and is an expected consequence of CRHR2 receptor activation, which triggers cAMP-PKA signaling (1, 2). The fact that UCn2 gene transfer did not increase glucose disposal in CRHR2-deleted mice rendered insulin resistant by HFD (Figure 4B) supports this idea. A previous study (9) found no change in fasting blood glucose after 4 weeks of HFD in a different CRHR2-deleted line, which may reflect a difference in line or an inadequate duration of HFD (4 weeks vs. 8–18 weeks in the current study).

Metabolic effects. UCn2 activation of CRHR2 receptors in brain and the gastrointestinal tract can alter satiety and thereby reduce food consumption (3). Although we saw nonsignificant reductions in food consumption (5%; P = 0.10), there was a 34% reduction in weight gain (P = 0.002) in UCn2 mice on HFD (Figure 7A). However, db/db mice showed similar increases in glucose disposal after UCn2 gene transfer but no change in food consumption or weight gain, indicating that UCn2’s effects on glucose disposal are not due to reduced weight alone. Metabolic studies of mice fed HFD for 16–17 weeks indicated that UCn2 gene transfer did not increase total activity, heat generation, or the rates of oxygen consumption or carbon dioxide production (Table 3). The biological importance of a 2.4% increase in respiratory exchange ratio following UCn2 gene transfer is unknown, although it indicates a small increase in metabolism of carbohydrate vs. fat.

Gross malabsorption was not evident, but to evaluate this possibility, we performed bomb calorimetric studies on stool from HFD-fed mice. We found that UCn2 gene transfer had no effect on energy loss in stool, which makes malabsorption unlikely. We then evaluated feed efficiency, a measure of weight gained per food consumed (10), and documented that UCn2 gene transfer was associated with a 50% reduction in feed efficiency (P < 0.014). The values for feed efficiency in mice on HFD (0.06 ± 0.01 g/g) are in line with previous reports, where a mean feed efficiency of 0.06 g/g was reported (10).

A treatment for T2DM that increases insulin sensitivity and glucose disposal while attenuating weight gain and reducing fatty infiltration of liver would be a welcome addition to current clinical therapy. Reduced fatty infiltration of the liver appeared to be independent of weight gain; it also was seen after UCn2 gene transfer in db/db mice, despite no group difference in weight. HFD mice that received UCn2 gene transfer had reduced hepatic triglyceride content and reduced liver weight.

Previous studies. Elevation in plasma UCn2 increased insulin sensitivity, an unanticipated and previously unreported phenomenon. Indeed, UCn2 deletion was reported to increase skeletal muscle glucose clearance (11). However, deletion of a gene is not the opposite of its expression vis-à-vis overall physiological integration. For example, this finding, which seems to contradict our studies, may reflect centrally mediated effects. In a transgenic line with general UCn3 overexpression, increased glucose clearance in HFD-fed mice was reported (12). However, these UCn3-expressing mice, unlike the mice that received UCn2 gene transfer in the present study, showed no change in insulin sensitivity. The mechanism for increased glucose clearance in the UCn3 transgenic line may relate to phenotypic features of the line, which included increased skeletal muscle mass and body weight. In addition, that study found a 44-fold increase in brain UCn3, which confounds data interpretation because of central effects of UCn3. In our study, UCn2 expression was not increased in the brain (Table 2).

Gene and cell therapy. The potential safety of i.v. delivery of an AAV8 vector was demonstrated in an early-phase gene transfer clinical trial in patients with hemophilia B (14). Although we can find no previous reports using gene transfer of an insulin-sensitizing peptide to treat insulin resistance or T2DM, there have been reports of other therapies. For example, islet-cell transplantation has been successful clinically (15), although donors are difficult to procure and rejection of donor islets has been a problem. An early-phase clinical trial showed that polyclonal regulatory T cell immunotherapy might be effective in treating type 1 diabetes (16). Gene transfer has been successful in preclinical models of diabetes. For example, insulin and glucokinase gene transfer (17) and gene transfer of both the NK1 fragment of hepatocyte growth factor (HGF/NK1) and glucagon-like peptide-1 (GLP-1), which appears to promote islet generation (18), have been successful in preclinical studies. However, these therapies have focused on insulin deficiency — type 1 diabetes and late-stage T2DM. In contrast, the current studies focused on earlier-stage insulin resistance using a transgene that increases insulin sensitivity.

Cardiac effects of UCn2. We have demonstrated that UCn2 gene transfer in normal mice has beneficial effects on LV contractile function through augmentation of Ca2+ handling (5). The safety and efficacy of UCn2 peptide infusion has been confirmed in large-animal models of HF (19) and in patients with HF (4). A clinical HF study using infusion of an UCn2-related peptide, stresscopin, found similar results (7). We also recently showed that UCn2 gene transfer increases function of the failing heart in mice (20). T2DM is commonly present in patients with HF. In patients with both HF and T2DM, UCn2 gene transfer could, in theory, resolve insulin resistance and increase LV function concurrently.

In clinical trials, UCn2 peptide infusion has been bedeviled with hypotension and reflex tachycardia. In the present study, we saw no group differences in mean daily heart rate assessed by continuous telemetry or in basal blood pressure assessed by tail cuff (Table 1). This may reflect tachyphylaxis to vasodilation associated with long duration and gradual-onset UCn2 exposure. In contrast, the effects of UCn2 gene transfer on blood glucose persisted for the duration of testing (16–17 weeks) without decrement in degree of effect. The absence of basal hypotension and tachycardia is reassuring vis-à-vis prospects for future clinical trials.

Translation to clinical application. AAV8.UCn2 dose-response data (Figures 8, A–D) indicate that a dose of 5 × 1010 gc (1.9 × 1012 gc/kg) is associated with a 44% reduction in AUC, and a dose of 1.6 × 1010 gc (6 × 1011 gc/kg) with an 18% reduction in AUC (Figure 8, C and D). Previous studies have shown that a 20% reduction in AUC is efficacious in treating clinical T2DM (21), suggesting that a dose of 6 × 1011 gc/kg, 30-fold lower than the highest dose used in the present study, may be effective in clinical settings. This would limit adverse effects that may be associated with higher doses of AAV8. These doses (1.9 × 1012 gc/kg and 6 × 1011 gc/kg) are similar to the highest 2 doses of an AAV8 vector encoding Factor IX used safely in a gene-transfer trial in human subjects with hemophilia B (2 × 1012 gc/kg, and 6 × 1011 gc/kg, i.v.) (14).

Clinical implications. Diabetes was reported to affect 12%–14% of the US adult population in 2011–2012 (22), 95% of whom have T2DM. T2DM is a major risk factor for stroke, neuropathy, kidney failure, blindness, myocardial infarction, and HF. Few diseases affect so many organs or are as prevalent. The discovery and development of more effective therapies is imperative. Intravenous delivery of an AAV vector encoding UCn2 provides numerous possible advantages over oral T2DM agents and insulin: (a) the insulin-sensitizing effect of UCn2 gene transfer would be anticipated to preserve β-cell function, a goal in the management of patients with early T2DM; (b) some oral T2DM agents, including thiazolidinediones, appear to be hazardous in patients with coronary artery disease or HF (23), but in contrast, UCn2 has beneficial effects on the heart (2, 4, 5, 19, 20); (c) repeated insulin injections or frequent oral medications are a nuisance for many patients — UCn2 gene transfer would circumvent this with a one-time treatment; (d) insulin and some oral T2DM agents are associated with weight gain, but in contrast, UCn2 gene transfer is associated with reduced weight gain in mice fed HFD (Figure 7A); and (e) UCn2 gene transfer reduced fatty infiltration of the liver in both models of insulin resistance (Figures 7, B and C). Nonalcoholic fatty liver disease is a rapidly increasing problem, particularly among patients with T2DM and metabolic syndrome, and is a common cause for liver transplantation (24). So this effect of UCn2 gene transfer in the setting of insulin resistance has important clinical implications.

Limitations. The precise molecular pathways beyond Glut4 translocation that explain the favorable effects of UCn2 gene transfer on glucose disposal will require additional studies. Elevation of UCn2, an endogenous peptide hormone, could potentially have adverse consequences in humans that are not apparent in mice. However, T2DM is a life-threatening disease, and despite many currently available medical options, few appear to effectively reduce its morbidity and mortality. The proposed therapy would serve an unmet medical need. Studies in nonhuman primates with T2DM will be initiated soon, and if insulin sensitivity and glucose disposal are safely increased in these studies, we hope to initiate a clinical trial.

An AAV8 vector encoding murine UCn2 with a chicken β-actin (CBA) promoter (Figure 1A) was produced as detailed previously (5). Plasmid pRep2/Cap8 was obtained from the University of Pennsylvania Vector Core.

Animal use

The numbers of animals used in each set of experiments, their weights, ages, sex, and animals that died or had unusable data are outlined in Table 5. There were 203 mice used (obtained from The Jackson Laboratory), ranging in age from 5–16 weeks, weighing 27.7–35.6 grams, depending on the protocol. All but 12 animals were male. Thirteen mice were excluded: 8 due to unusable data (determination made prior to unblinding) and 5 due to death during anesthesia or surgery — 4 prior to vector delivery (Table 5).

To determine the effect of UCn2 gene transfer on glucose disposal, we used HFD to induce insulin resistance and hyperglycemia in normal mice. Mice were provided (ad libitum) a cereal-based diet (Harlan Teklad Lab) for 5 weeks and then switched to a HFD (60 kcal%; Research Diets) for durations and group sizes indicated in individual experiments. Mice received i.v. AAV8.UCn2 (5 × 1011 gc) or saline either at the initiation of HFD or 8 weeks later. Mice were housed (20oC–21oC) with lights off from 6 pm to 6 am daily. Food consumption and body weight were recorded weekly. A second group of normal mice was fed HFD for 16 weeks. After week 8, they received i.v. injection of: saline, AAV8.EGFP (5 × 1011 gc), or AAV8.UCn2 in 1 of 5 doses from 5 × 109 gc to 5 × 1011 gc in half-log increments. These data were used to determine the relationship between AAV8.UCn2 dose and plasma UCn2 concentration and glucose disposal via glucose tolerance testing.

Under anesthesia (1.5% isoflurane via nose cone) a small incision was made on the neck to expose the jugular vein for i.v. delivery. Mice were injected with AAV8.UCn2 (in 50 μl PBS) or an equivalent volume of saline (control).

These studies were performed on HFD-fed mice (8 weeks old; Table 5). Upon receipt, they were continued on HFD and received i.v. UCn2 gene transfer (5 × 1011 gc; n = 9) or i.v. saline (n = 9) within 1 week. The 2 groups underwent clamp studies 17 ± 1 weeks after gene transfer, having been sustained on HFD continuously. Clamps were conducted in weight-matched conscious mice after a 6-hour fast as previously described (26). Mice were anesthetized using ketamine (100 mg/kg, Zoetis), acepromazine (3 mg/kg, Vet One), and xylazine (10 mg/kg, Aveco) and jugular vein was cannulated 4 days before clamp measurement. On the day of the clamp, body weight was recorded and blood glucose measured (6 hr fasting). Mice then were placed in a Lucite restrainer (Braintree Scientific) and blood samples obtained (–60 min) for plasma insulin concentration. Equilibrating [3-3H] D-glucose tracer solution (PerkinElmer; 41.6 μCi 3H/ml) was then infused (2 μl/min i.v., 60 min). At the end of the equilibration period (t = 0 min), two 15-μl blood samples were obtained and deproteinized using 125 μl ZnSO4 (0.3 N) and 125 μl BaOH (0.3 N) for assessment of tracer-specific activity and basal glucose disposal rate. To clamp after the equilibration period, a cocktail containing 8% BSA, insulin (Humulin R, Lilly, 10.0 mU/kg/min), and tracer (41.6 μCi/ml) was infused at a constant rate (2.0 μl/min) along with a variable glucose infusion (50% dextrose, 454 mg/ml). Blood glucose was assessed using blood glucose meter and test strips every 10 min, and infusion rate was adjusted until steady-state blood glucose (120 ± 10 mg/dl) was achieved. The clamp was terminated when steady-state conditions were maintained for ≥ 20 minutes (~120 min), at which time two 15-μl blood samples were obtained for assessment of tracer-specific activity and insulin-stimulated glucose disposal rate (t = ~120 min). Blood (70 μl) was collected before and after the clamp for measurement of plasma insulin concentration.

CRHR2-deleted mice

To determine whether UCn2 effects on glucose disposal were mediated through its cognate receptor, CRHR2, UCn2 gene transfer was performed in CRHR2-deleted mice 8 weeks after initiation of HFD, which was continued for a total of 17 weeks (9 weeks after gene transfer). CRHR2+/– × CRHR2+/– mice and CRHR2–/– × CRHR2–/– (C57BL/6 background) were bred to obtain CRHR2+/+ (WT) and CRHR2–/– mice (27). Mice were housed in a temperature and light-controlled room (22°C–24°C; 12 hr light/12 hr dark) and were bred at UCSF’s centralized vivarium.

To determine if UCn2 gene transfer affected absorption of food, 12 mice were placed on HFD (33 weeks). At week 13, 6 of 12 received i.v. UCn2 gene transfer (5 × 1011 gc). The energy content in fecal samples was measured by Kinetica Inc. Stool samples (stored at –20°C) were dried in a convection oven (100°C, 6 hr). Energy content for each stool specimen was determined from the heat of combustion measured with an oxygen bomb calorimeter. Testing was conducted according to standard guidelines (28).

Metabolic Studies

The Comprehensive Laboratory Animal Monitoring System (CLAMS, Columbus Instruments) was used to assess oxygen consumption and activity level in 12 normal mice fed HFD for 16 weeks. Six of 12 mice received i.v. AAV8.UCn2 (5 × 1011 gc) midway through the 16-week HFD period. Mice were placed in standard metabolic cages for 5 days while measurements were continuously acquired. Data from 5 light/dark cycles following a 1.5-day acclimation period were used in the analysis.

A subset of mice fed HFD for 8 weeks received i.v. UCn2 gene transfer (5 × 1011 gc, n = 8) or saline (n = 8), were continued 8 additional weeks on HFD, and were then killed. Samples of liver and transmural sections of the LV free wall were formalin fixed and paraffin imbedded. Five micron sections were mounted and counterstained with H&E and with Masson’s trichrome and examined for fibrosis and inflammation. To quantify fatty infiltration of liver, slides were scanned (NanoZoomer 2.0HT Slide Scanner), planimetered, and reported as percentage of total area.

Hepatic and skeletal muscle triglyceride content

Hepatic and skeletal muscle triglyceride content was determined using a colorimetric/fluorometric kit (Biovision Inc.). Liver and skeletal muscle samples (100 mg) were homogenized in 1 ml of water containing 5% NP-40 using Tissuemiser (Thermo Fisher Scientific). Lysates were heated (100°C, 5 min) and cooled (25°C) twice. After centrifugation (15,000 g, 2 min) the supernatant was assayed for triglyceride content, which was reported as nmol per mg of liver or skeletal muscle (wet weight).

Necropsy

Body, liver, lung, and LV weight (including interventricular septum), as well as tibial length, were recorded. A short axis midwall LV ring, a short-axis sample of skeletal muscle at mid-calf level, and samples of liver were obtained; portions were quickly frozen in liquid nitrogen and stored at −80°C or fixed in formalin and embedded in paraffin.

Statistics

Data represent mean ± SEM; group differences were tested for significance using Student’s t test (unpaired, 2-tailed) or repeated measures ANOVA. In glucose tolerance tests, the trapezoidal rule was applied to determine AUC. The null hypothesis was rejected when P < 0.05. Analyses were performed using GraphPad Prism (GraphPad Software Inc.). Those collecting and analyzing data obtained from physiological experiments were blinded to group identity.

Study approval

The Guide for the Care and Use of Laboratory Animals. (National Academies Press. 2011.) was followed, and the Animal Use and Care Committees of the VA San Diego Healthcare System and the UCSF approved the studies.

We thank Ruoying Tang for her technical expertise and the UCSD Animal Care Program Phenotyping Core for performing the metabolic cage experiments. We thank Tamsin Lisa Kelly for reviewing the manuscript and providing helpful criticisms. This work was supported by NIH grants (P01 HL66941, HL088426, and DK080787); an NHLBI Gene Therapy Resource Program grant (HHSN268201200041C); VA Merit grant (1101BX001515); and UCSD School of Medicine Light Microscopy Facility (grant NS047101) for imaging.